Research in the investigator's laboratory has focused on the two dsRNA-dependent IFN- inducible 2'5'oligoA synthetase/RNase L and PKR pathways. They have reported a statistically significant dysregulation in which the 2'5'A synthetase is present in its activated form, 2'5'A levels are elevated, RNase L is upregulated and the expression of PKR is downregulated. Recent data suggest additional differences unique to peripheral blood mononuclear cells (PBMC) from individuals with CFS compared to normal controls. Specifically, these findings in CFS PBMC are: 1) a 36kDa 2'5'A binding protein which is recognized by an RNase L polyclonal antibody; 2) a 90% decrease in cellular actin expression and 3) a pronounced change in total protein profiles. These data place the investigator in a unique position to explore the 2'5'A synthetase/RNase L and PKR systems in CFS. The proposed research will examine the 2'5'A synthetase and PKR pathways in an in vitro model and PBMC from a cohort of CFS patients and two control populations, in association with clinical symptomatology. The working hypothesis is that the characteristic signs and symptoms of CFS are associated with the dysregulation of the 2'5'A synthetase/RNase L and PKR pathways. The project will address: 1) the expression and activities of the four isoforms of 2'5'A synthetase; 2) the activators of 2'5'A synthetase; 3) the intracellular concentration, oligomer distribution and stability of 2'5'A; 4) the identification and characterization of the newly-discovered 36 kDa 2'5'A binding protein that is immunoreactive with RNase L antibody; 5) synthetic and natural routes for the inhibition of the upregulated RNase L in CFS; 6) the ultimate implications of the upregulated RNase L activity and 7) potential mechanisms for the decreased expression of PKR. The studies in the application are suggested to contribute to development of potential rational therapies for CFS.